7-deazaguanines as immunomodulators

ABSTRACT

The present invention is various 7-deazaguanines having activity as immunomodulators. Also included are pharmaceutical compositions and methods of use thereof.

This is a Divisional of U.S. application Ser. No. 07/354,312, filed Mar.13, 1989 now U.S. Pat. No. 4,921,858; which is a continuation ofPCT/US87/02727 filed Oct. 19, 1987; which is a continuation of U.S.application Ser. No. 07/086,231, filed Aug. 20, 1987 now abandoned;which is a continuation-in-part of U.S. application Ser. No. 06/923,521,filed Oct. 24, 1986 now abandoned.

BACKGROUND OF THE INVENTION

The pyrrolo[2,3-d]pyrimidin-4-ones of the following formula 2, 3 and 4##STR1## are known. R. K. Robins, et al, synthesized the compounds offormula 2 and 3 as reported in J. Het. Chem., 1964, 34, but gave nobiological activity for either compound. M. Legraverend, et al, reportedthe synthesis of the compounds of formula 3 and 4 in TetrahedronLetters, 1985, 2001, but again gave no biological activity for eithercompound.

Of lesser interest the following references provide a background inwhich a 7-(substituted phenyl)pyrrolo[2,3-d]pyrimidin-4-one having amethyl at each of the five (5) and six (6) positions for treating CNSillnesses or inflammations is disclosed generically in U.S. Pat. No.4,229,453. Similarly, 4-mercapto-7-(phenyl substituted orunsubstituted)pyrrollo [2,3-d]pyrimidine derivatives requiring an alkylor phenyl at the five (5) and six (6) positions are disclosed in German3145287 (Derwent Abstract No. 49344 K/21). Other pyrrolo[2,3-d]pyrimidin-4-one or thione, distinguished by having varioussubstituents at the five (5) position, are found in U.S. Pat. Nos.4,435,570 and 4,140,851; European publications 160,910 (Derwent AbstractNo. 85-284574/46); 89,055 (corresponding to U.S. Pat. No. 4,571,423);119,591 (Derwent Abstract No. 84-238735/39); 79,447 (corresponding toU.S. Pat. No. 4,435,569); German 3,306-390 (Derwent Abstract No. 39438E/20); German 3145287 (Derwent Abstract No. 49344 K/21); British PatentNo. 981,458 (Derwent Abstract No. 15,454); Japanese J6 0204,788 (DerwentAbstract No. 85/298810/48); and Japanese J5 9036615 (Derwent AbstractNo. 84-086061/84).

Finally, hydroxy and mercapto analogs of the antibiotic sparsomycin Aare pyrrolo[2,3-d]pyrimidin-4-one or thione having a sugar moiety in theseven (7) position are disclosed by Upjohn in Netherlands 6,407,785(Derwent Abstract No. 15,466) and similarly by Warner Lambert inEuropean publication 57,548 (Derwent Abstract No. 68572 E/33).

Copending U.S. application Ser. Nos. 336,585, now U.S. Pat. No.4,923,872, which is a continuation of U.S. Ser. No. 117,352 filed Nov.12, 1987, abandoned, which is a continuation-in-part of U.S. Ser. No.059,419 filed June 18, 1987, abandoned, which is a continuation-in-partof U.S. Ser. No. 900,486 filed Aug. 26, 1986, abandoned and U.S. Ser.No. 767,202 filed Aug. 22, 1985, now U.S. Pat. No. 4,772,606, which is acontinuation-in-part of U.S. Ser. No. 660,152 filed Oct. 12, 1984, nowabandoned, disclose similar activity as now found in the presentinvention for different ring systems.

SUMMARY OF THE INVENTION

The present invention relates to a compound of the formula (I) ##STR2##wherein R₆ is OH or SH, R₂ is hydrogen or NH₂, R₇ and R₈ areindependently hydrogen or NH₂ with the proviso that both cannot be NH₂at once, n is an integer of from one through four, Ar is (i) phenylunsubstituted or substituted by halogen, trifluoromethyl, alkyl of oneto four carbon atoms, hydroxy, or alkoxy of from one to four carbonatoms, (ii) 2- or 3-thienyl, or (iii) 2- or 3-furanyl with the provisothat when R₆ is OH, and R₂ is H₂ N, and R₇ and R₈ are both hydrogen thenAr cannot be unsubstituted phenyl; or a pharmaceutically acceptable baseor acid addition salt thereof.

The present invention also includes methods of manufacturing and novelintermediates therein, and a pharmaceutical composition for treatingautoimmune diseases such as arthritis, systemic lupus erythematosus,inflammatory bowel diseases, juvenile diabetes, myasthenia gravis,multiple sclerosis, gout and gouty arthritis, as well as psoriasis,viral infections and cancer, or rejection of transplantation, comprisingan anti-psoriatic, immunomodulator or antirejection effective amountsuch as an advantageously cytotoxic to T-cell amount, of a compound ofthe formula (I) ##STR3## or a pharmaceutically acceptable base or acidaddition salt thereof wherein R₆ is OH or SH, R₂ is hydrogen or NH₂, R₇and R₈ are independently hydrogen and NH₂ with the proviso that bothcannot be NH₂ at once, n is an integer of from one to four, Ar is (i)phenyl unsubstituted or substituted by halogen, trifluoromethyl, alkylof one to four carbon atoms, hydroxy, alkoxy of from one to four carbonatoms, (ii) 2- or 3-thienyl, or (iii) 2- or 3-furanyl with apharmaceutically acceptable carrier. Thus, the invention is also amethod of treating psoriasis, an autoimmune disease, such as is listedabove, or rejection of transplantation comprising administering to ahost, such as a mammal including a human, suffering from psoriasis, theautoimmune disease or rejection of transplantation comprisingadministering an effective amount; i.e. an amount advantageouslyaffecting T-cells by toxicity thereto, of a pharmaceutical compositionof the formula I as defined above in unit dosage form. It is understood,an ordinarily skilled physician would begin treatment with a less thaneffective amount and increase the dose until the desired effect isobtained exercising care to administer an amount less than the amounttoxic to the host of the disease.

The novel intermediates of the present invention are compounds offormula (X) ##STR4##

The method of manufacture of the present invention is a novel processfor the preparation of a compound of the formula I as defined above;which comprises treating a compound of the formula (X) ##STR5## with acompound of the formula (V) ##STR6## wherein Ar and n are as definedabove and then treating with an acid to obtain the compound of formula Iwherein R₆ is oxygen and R₈ is NH₂ and alternatively, if desired,further deaminating by known methods or treating also by known methods,to obtain a compound of formula I wherein R₆ is sulfur and then, ifdesired, deaminating.

The compound of formula X is prepared by treating a compound of theformula (IX) ##STR7## with a cyanation agent, such asO,N-bistrifluoroacetylhydroxylamine, in the presence of a base, such aspyridine to obtain the compound of formula X.

The above preparations use standard synthetic techniques or techniquesas shown or similar to those as shown in the examples hereinafter. Thestarting materials for the preparation are readily available, known orcan be prepared by known methods.

The methods of manufacture for the compounds of the present inventionare summarized in the following Schemes I and II. ##STR8##

Under certain circumstances it may be necessary to protect either the Nor O of intermediates in the above noted process with suitableprotecting groups which are known. Introduction and removal of suchsuitable oxygen and nitrogen protecting groups are well-known in the artof organic chemistry; see for example, (1) "Protective Groups in OrganicChemistry," J. F. W. McOmie, ed., (New York, 1973), pp 43ff, 95ff; (2)J. F. W. McOmie, Advances in Organic Chemistry, Vol. 3, 191-281 (1963);(3) R. A. Borssonas, Advances in Organic Chemistry, Vol. 3, 159-190(1963); and (4) J. F. W. McOmie, Chem. & Ind., 603 (1979).

Examples of suitable oxygen protecting groups are benzyl,t-butyldimethylsilyl, methyl, isopropyl, ethyl, tertiary butyl,ethoxyethyl, and the like. Protection of an N-H containing moiety isnecessary for some of the processes described herein for the preparationof compounds of this invention. Suitable nitrogen protecting groups arebenzyl, triphenylmethyl, trialkylsilyl, trichloroethylcarbamate,trichloroethoxycarbonyl, vinyloxycarbamate, and the like.

Under certain circumstances it is necessary to protect two differentoxygens with dissimilar protecting groups such that one can beselectively removed while leaving the other in place. The benzyl andt-butyldimethylsilyl groups are used in this way; either is removable inthe presence of the other, benzyl being removed by catalytichydrogenolysis, and t-butyldimethylsilyl being removed by reaction with,for example, tetra-n-butylammonium fluoride.

In the process described herein for the preparation of compounds of thisinvention the requirements for protective groups are generally wellrecognized by one skilled in the art of organic chemistry, andaccordingly the use of appropriate protecting groups is necessarilyimplied by the processes of the charts herein, although not expresslyillustrated.

The products of the reactions described herein are isolated byconventional means such as extraction, distillation, chromatography, andthe like.

The salts of compounds of formula I described above are prepared byreacting the appropriate base with stoichometric equivalent of the acidcompounds of formula I to obtain pharmacologically acceptable saltsthereof.

The compounds of this invention may also exist in hydrated or solvatedforms.

DETAILED DESCRIPTION

The compounds of formula I of the present invention exist in tautomericforms as purines or guanines as illustrated below. Both forms areincluded as part of the invention and are indiscriminately described inthe specification. ##STR9##

The term "alkyl of one to four carbon atoms" means a straight orbranched hydrocarbon chain up to four carbon atoms such as, for example,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl ortertiary butyl. Alkoxy of one to four carbon atoms includes methoxy,ethoxy, propoxy, butoxy and isomers thereof. Halogen is fluorine,chlorine, bromine, or iodine.

The compounds of formula I are useful both in the free base form, in theform of base salts where possible, and in the form of acid additionsalts. The three forms are within the scope of the invention. Inpractice, use of the salt form amounts to use of the base form.Appropriate pharmaceutically acceptable salts within the scope of theinvention are those derived from mineral acids such as hydrochloric acidand sulfuric acid, and organic acids such as methanesulfonic acid,benzenesulfonic acid, p-toluenesulfonate, and the like, respectively, orthose derived from bases such as suitable organic and inorganic bases.Examples of suitable inorganic bases for the formation of salts ofcompounds of this invention include the hydroxides of ammonia, sodium,lithium, potassium, calcium, magnesium, aluminum, zinc, and the like.

Salts may also be formed with suitable organic bases. Bases suitable forthe formation of pharmaceutically acceptable base addition salts withcompounds of the present invention include organic bases which arenontoxic and strong enough to form such salts. These organic bases forma class whose limits are readily understood by those skilled in the art.Merely for purposes of illustration, the class may be said to includemono-, di-, and trialkylamines, such as methylamine, dimethylamine, andtriethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-,and triethanolamine; amino acids such as arginine, and lysine;guanidine; N-methylglucamine; L-glutamine; N-methylpiperazine;morpholine; ethylenediamine; N-benzylphenethylamine;tri(hydroxymethyl)aminomethane; and the like. (See for example,"Pharmaceutical Salts," J. Pharm. Sci. (1977) 66(1):1-19.)

The acid addition salts of said basic compounds are prepared either bydissolving the free base of compound I in aqueous or aqueous alcoholsolution or other suitable solvents containing the appropriate acid orbase and isolating the salt by evaporating the solution, or by reactingthe free base of compound I with an acid as well as reacting compound Ihaving an acid group thereon with a base such that the reactions are inan organic solvent, in which case the salt separates directly or can beobtained by concentration of the solution.

A preferred embodiment of the present invention is a compound of formulaI wherein R₆ is OH or SH; R₂ and R₈ are NH₂, n is one, and Ar is 2- or3-thienyl. A more preferred embodiment is2-amino-7-(2-thienylmethyl)-4-pyrrolo[2,3-d)]pyrimidone.

The compositions having compounds of the formula I of the presentinvention are shown to exhibit significant enzyme inhibition activityand cytotoxicity activity. In the purine nucleoside phosphorylase(PNP-4) enzyme assay, an IC₅₀ is achieved at a dose of 1.0 micromoles ona selected compound of the present invention. PNP-4 activity for thecompound of formula I is measured radiochemically by measuring theformation of [¹⁴ -C]hypoxanthine from [¹⁴ -C]inosine [Biomedicine, 33,39 (1980)] using human erythrocyte as the enzyme source.

It is known that an in vivo inhibition of purine nucleosidephosphorylase (HPLC-1) enzyme assay may also be used essentially asdisclosed in the Annals of New York Academy of Sciences, Volume 451,Page 313 (1985) to further show the activity for compositions of thecompounds of formula I of the present invention. The present inventioncompositions also are generally shown by a standard test (HTBA-1)[Science, 214, 1137, (1981)]to be selectively cytotoxic for T-cells inthe presence of 2'-deoxyguanosine at a similar concentration range andnontoxic to B-cell in the presence of the same amount of2'-deoxyguanosine by the compound of Example 2, thus demonstratingutility for the compounds of formula I in pharmaceutical compositions asdescribed herein. Since PNP inhibition and removal of T-cells ormodulation of T-cells are known to be characteristics of compoundsbeneficial in the treatment of psoriasis, rejection phenomenon intransplantation, and autoimmune diseases, the present inventioncompositions of compounds being selectively cytotoxic to T-cells andbeing PNP inhibitors will, therefore, also be useful in such treatment.For example, 8-Aminoguanosine, a known PNP-inhibitor, has been shown tobe efficacious for inhibiting rejection of skin graft in dogs [J. B.Benear, et al, Transplantation, 1986, 41:274]. Clinically it has beenshown that modulation and/or removal of T-cells by thoracic ductdrainage, lymphapheresis or total lymphoid irradiation gave partial tocomplete relief from rheumatoid arthritis in patients who were totallyrefractory to other forms of therapy (A. Tanay, et al, Arthritis andRheumatism, Vol. 30, No. 1, p. 1 (1987). S. Strober, et al, Annual ofInternal Medicine, V-102, No. 4, 441-449 (1985); H. G. Nusslein, et al,Arthritis and Rheumatism, V-28, No. 11, 1205-1210 (1985); E. Brahn, etal, ibid, V-27, No. 5, 481-487 (1984), and J. Karsh, et al, ibid, V-24,No. 7, 867-873 (1981)). Cyclosporin A, a T-cell modulator, showedbeneficial effects in the treatment of juvenile diabetes. (A. Assan, etal, The Lancet, January 12, p. 67 (1985).) Additionally, cyclosporin Ais presently the drug of choice for the prevention of transplantrejection, (R. M. Merion, et al, New Eng. J. Med., (1984) 148). Morerecently, cyclosporin A is shown to be useful to treat psoriasis.Further, it is suggested the cyclosporin therapy is shown to markedlyreduce activated T-cells in psoriatic lesions. Therefore, it isreasonable to believe the basis of the successful treatment of psoriasisis modulation of T-cell activity as shown by compounds in the presentinvention composition. (See C. N. Ellis, et al, JAMA, V-256, No. 22,Dec. 12, 1986, pp. 3110-3116.) Finally, cyclosporin A is shown to beefficacious in rheumatoid arthritis. (M. E. Weinblatt, et al, Arthitisand Rheumatism, V-30, No. 1, pp. 11-17 (January, 1987); 0. Forre, et al,Arthritis and Rheumatism, V-30, No. 1, pp. 88-92 (January, 1987); M.Dougados, et al, Arthritis and Rheumatism, Vol. 30, No. 1, pp. 83-87(January, 1987).

Representative examples from the present invention are shown in thefollowing activity table to provide the activity discussed above.

                  ACTIVITY TABLE                                                  ______________________________________                                         ##STR10##                                                                                                       HTBA-1                                                                PNP-4   T-Cell + 10 μM                          Number R.sub.7                                                                              R.sub.8                                                                              Ar    IC.sub.50 (μM)                                                                     2'-d Gua; IC.sub.50 μM                  ______________________________________                                        2      H      H      2-Th  1.0     2.3                                        ______________________________________                                         Th = Thiophene                                                           

In vivo studies based on the above noted disclosures may be used todetermine activity in the particular disease states noted.

Since T-cells play a central role in immune response, use of thecompounds of the invention is contemplated for the immunoregulation toprevent rejection in transplantation or in the treatment of psoriasisand in the treatment of autoimmune disease such as rheumatoid arthritis,systemic lupus erythrematosus, inflammatory bowel disease, multiplesclerosis, myasthemia gravis, gout or gouty arthritis, juvenilediabetes, cancer, and viral diseases. The present invention thusincludes compositions containing a compound of formula I in treatingrejection of transplantation or disease such as psoriasis in humans orautoimmune disease characterized by abnormal immune response in primatesor humans. According to this aspect of the invention, the properties ofthe compounds of the invention are utilized by administering to awarmblooded animal an effective amount of a pharmaceutical compositioncontaining as the active ingredient at least about 0.1 percent byweight, based on the total weight of the composition of at least onesuch compound of the invention.

Pharmaceutical compositions of the invention can be formulated in anysuitable way, preferably with an inert carrier for administrationorally, parenterally, ophthalmically, topically, or by suppository.

For example, the compounds of the present invention are formulated intodosage forms such as tablets or syrups by blending with an inertpharmaceutical carrier such as lactose or simple syrup by methodswell-known in the art. For injectable dosage forms, they are formulatedwith vehicles such as water, propylene glycol, peanut oil, sesame oil,and the like. In these dosage forms, the active ingredient is from about0.05 grams to 0.5 grams per dosage unit.

The present invention is further illustrated by way of the followingexamples.

EXAMPLE 1 2-Amino-6-[(2-thienylmethyl)amino]-4-pyrimidinol

2-Amino-6-chloro-4-pyrimidinol, monohydrate (85%, 100 g, 0.5197 mol) wassuspended in methoxyethanol (700 ml) and 2-thienylmethylamine (96%, 61.3g, 0.5197 mol) was added to the suspension. The mixture was heated underreflux for two hours and then 73 ml (d=0.726; 0.52 mol) of triethylaminewas added and the refluxing continued for an additional 18 hours. Thereaction mixture was poured into ice water (1000 ml), acidified withacetic acid (ph 4.0) and the precipitated solid was filtered, washed,and dried. Yield: 110 g (72.6%). This was used in the next step withoutfurther purification.

EXAMPLE 2 2-Amino-7-(2-thienylmethyl)-4-pyrrolo[2,3-d]pyrimidones

Chloroacetaldehyde dimethyl acetal (14 ml) was added to water (50 ml)and concentrated HCl (2.0 ml). The mixture was heated at refluxtemperature for 30 minutes and then neutralized with sodium acetate (10g). The resulting solution was added in one portion to a mixture of2-amino-4-(2-thienylmethyl)-6-pyrimidone (10 g; 45 mmol), sodium acetate(5.0 g) and hot water (50 ml). The mixture was allowed to stir on asteam bath (80° C.) for 30 minutes, and the precipitated solid wasfiltered, washed with water, and dried in vacuo. The crude product wasdissolved in methanol and concentrated HCl and treated with charcoal toremove coloring matter. The product (3.2 g) thus obtained wasrecrystallized from methanol and 1 N HCl (100 ml) to give 1.68 g (13.5%)of the desired product as light brown solid, mp 243°-245° C. (dec).

2-Amino-4-chloro-6-[2-thienylmethyl)amino]-5-(2,2-diethoxyethyl)pyrimidine

A solution of 2-amino-4,6-dichloro-5-(2,2-diethoxyethyl) pyrimidine (M.Legraverend, et al, J. Med. Chem., 1985, 28:1477) (906 mg, 3.20 mmol) in40 ml of n-butanol containing Et₃ N (1 ml) and 2-thienylamine (425 mg,3.75 mmol) was heated at 100° C. for 48 hours. The reaction mixture wascooled to 25° C. and concentrated. The residue was cooled to 25° C. andconcentrated. The residue was purified by column chromatography oversilica gel and eluted with chloroform to give the desired product (1.045g) (91.5%) as a yellow oil.

2-Amino-4-chloro-7-(2-thienylmethyl)pyrrolo[2,3-d]pyrimidine

A suspension of 2-amino-4-chloro-6-[(2-thienylmethyl)amino]-5-(2,2-diethoxyethyl)pyrimidine (1.0 g, 2.80 mmol) in 65 ml of0.3N HCl and ethanol (2.25:1) was stirred at 25° C. for 24 hours. Thereaction mixture was neutralized with ammonium hydroxide solution andthe product collected by filtration. TLC analysis showed that thereaction was not complete, so, the product was resuspended in 50 ml of0.2N HCl and stirred for 48 hours. The reaction mixture was neutralizedwith NH₄ OH solution and concentrated. The residue was taken up in waterand then evaporated to dryness to give yellow solid (573 mg) (77.3%).This was used in the next step without further purification.

2-Amino-7-(2-thienylmethyl)-4-pyrrolo[2,3-d]pyrimidone

2-Amino-4-chloro-7-(2-thienylmethyl)pyrrolo[2,3-d]pyrimidine (563 mg,2.10 mmol) was suspended in 30 ml of 1 N HCl and ethanol (1:1) and themixture was heated at reflux for 8 hours. Removal of solvent gave aresidue which was chromatographed over silica gel and eluted with amixture of hexane-ethyl-acetate (10:1) to give 139 mg of a mixture of4-chloro and 4-ethoxy derivatives. So, it was dissolved in 30 ml of 3NHCl and the solution was heated to reflux for 2 hours and then allowedto cool. The precipitated solid was filtered and then recrystallizedfrom methanol - 1 N HCl (1:1) mixture to give 55 mg of the desiredproduct as hydrochloride salt, mp 235°-237° C. (dec).

We claim:
 1. A compound of the formula (I) ##STR11## wherein R₆ is OH orSH, R₂ is hydrogen or NH₂, R₇ and R₈ are independently hydrogen or NH₂with the proviso that both cannot be NH₂ at once, n is an integer offrom one through four, Ar is (i) phenyl unsubstituted or substituted byhalogen, trifluoromethyl, alkyl of one to four carbon atoms, hydroxy oralkoxy of from one to four carbon atoms, with the proviso that when R₇and R₈ are both hydrogen, R₆ is OH, R₂ is NH₂ and n is equal to one thenAr cannot be phenyl; or a pharmaceutically acceptable base or acidaddition salt thereof.
 2. A compound of claim 1 wherein R₇ is hydrogen.3. A compound of claim 2 wherein R₈ is hydrogen.
 4. A compound of claim2 wherein R₈ is amino.
 5. A pharmaceutical composition for treatingpsoriasis, autoimmune diseases or rejection of transplantationcomprising an antipsoriatic, antiautoimmune disease or antirejection oftransplantation effective amount of a compound of formula (I) ##STR12##or a pharmaceutically acceptable base or acid addition salt thereof;wherein R₆ is OH or SH, R₂ is hydrogen or NH₂, R₇ and R₈ areindependently hydrogen and NH₂ with the proviso that both cannot be NH₂at once, n is an integer of from one to four, Ar is (i) phenylunsubstituted or substituted by halogen, trifluoromethyl, alkyl of oneto four carbon atoms, hydroxy, alkoxy of from one to four carbon atoms,and a pharmaceutically acceptable carrier in unit dosage form.
 6. Amethod for treating autoimmune disease or rejection of transplantationwhich comprises administering a composition of claim 5 in unit dosageform.